Glycosylation of secretory proteins and the like is one of the important processes in the post-translational modification and controlled by various enzymes in the endoplasmic reticulum and the Goldi apparatus. Sugar chains attached to proteins by glycosylation consist of mannose (Man), galactose (Gal), N-acetylglucosamine and the like. Sugar chains attached to proteins are classified into two groups: N-linked sugar chains linked to the amide nitrogen atom in an asparagine residue and O-linked sugar chains linked to the hydroxyl oxygen atom in a serine or threonine residue. These sugar chains are said to stabilize proteins and have influences on the interaction between proteins and cells. Therefore, development of techniques for production of N-glycosylated proteins has been attempted for production of medicinal products (Patent Documents 1 and 2).
While various findings have been accumulated about N-glycosylation, little is known about O-glycosylation. In living organisms, O-glycosylated proteins are usually produced with sugar chains having various structures, and for production of medicinal products, control of sugar chain structure is important to produce proteins with sugar chains having specific structures. Therefore, techniques for production of proteins having O-linked sugar chains with specific structures with high productivity are demanded.
For production of desired proteins with high productivity, transformants carrying genes encoding heterologous proteins (which are not inherently produced by the host) introduced by genetic engineering are widely used. For production of eukaryotic proteins, eukaryotic microorganisms are considered as the best host, and mostly yeasts are used because they do not contain substances harmful to humans. Among them, the fission yeast Schizosaccharomyces pombe (hereinafter referred to as S. pombe) is said to be similar to animal cells in cell cycle, the chromosomal structure, RNA splicing and the like, as compared with budding yeasts such as Schizosaccharomyces cerevisiae (hereinafter referred to as S. cerevisiae) and is considered to produce proteins which have gone through post-translational modifications close to those in animal cells.
O-glycosylation in S. cerevisiae is initiated by attachment of mannose to oxygen atoms in serine or threonine residues of proteins catalyzed by O-mannosyltransferase encoded by the PMT gene family (Non-Patent Documents 1 and 2). The subsequent elongation of sugar chains involves α1,2-mannosyltransferase encoded by the KTR gene family and a1,3-mannosyltransferase encoded by the MNN1 gene family (Non-Patent Document 3).
However, the genes involved in elongation of O-linked sugar chains have not been identified.
For production of heterologous proteins in S. pombe, promoters, secretion signal genes, multicloning vectors functional in S. pombe have been developed. However, because little is known about genes involved in modification of O-linked sugar chains in S. pombe, it has been impossible to control the structure of O-linked sugar chains.
Under these circumstances, methods for producing heterologous proteins having specific O-linked sugar chains are demanded.